The present invention relates generally to the field of human genetics. Specifically, the present invention relates to methods and materials used to isolate and detect a human tumor suppressor gene, termed RASSF1 herein (sometimes also referred to as REH3P21). More specifically, the invention relates to the analysis of the RASSF1 gene and its use in the diagnosis of predisposition to cancer. The invention also relates to the prophylaxis and/or therapy of cancer associated with the RASSF1 gene. The invention further relates to the screening of drugs for cancer therapy. Finally, the invention relates to the screening of the RASSF1 gene for methylation, loss of heterozygosity or mutations, which are useful for diagnosing the predisposition to cancer.
The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are respectively grouped in the appended Lists of References.
Primary carcinoma of the lung is the leading cause of cancer death in the United States. Every year, more than 100,000 males and 50,000 females develop lung cancer, and most of them die within twelve months. There is a clinical need for a screening test which can detect lung cancer in its earliest stages because prompt treatment of localized disease improves the 5-year survival rate to 30% in males and 50% in females. However, most cases are not detected until local or metastatic growth causes symptoms, and prospective screening with frequent radiography and sputum cytology has not improved the survival rate in smoking males aged 45 years or older. Since early detection of lung cancer can potentially reduce mortality, researchers have investigated alternative diagnostic technologies such as identification of genes for determining risk for developing lung cancer.
Breast cancer is one of the most common malignancies in the world (Hunter, 2000). Only about 7% of breast cancer cases in the United States are thought to be due to the presence of an autosomal dominant susceptibility allele (Black and Weber, 1998). Two familial breast cancer genes (BRCA1 and BRCA2) have been identified (Ford and Easton, 1995). However mutations in these genes are relatively rare in the general population (Peto et al., 1999), and mutations are extremely uncommon in sporadic breast cancers (Futreal et al., 1994; Teng et al., 1996; Lancaster et al., 1996; Miki et al., 1996)). Alterations or changes in expression levels have been described for several other genes, including p53, E-cadherin, and HER-2/neu (Ingvarsson, 1999). It is very likely that additional genes contribute to inherited and sporadic breast cancer.
In general, both alleles of a tumor suppressor gene need to be inactivated by genetic alterations such as chromosomal deletions or loss-of-function mutations in the coding region of a gene (Knudson, 1971). As an alternative mechanism, epigenetic alterations of tumor suppressor genes may occur in human cancers resulting in gene inactivation. Recent studies have demonstrated that the CpG islands in the RB, p16, VHL, APC, and BRCA1 genes are frequently methylated in a variety of human cancers (Baylin et al., 1998; Jones and Laird, 1999; Herman and Baylin, 2000).
CpG islands that are hypermethylated in breast cancer are those of the genes coding for estrogen receptor (Ottaviano et al., 1994; Nass et al., 2000; Yoshida et al., 2000), retinoic acid receptor beta2 (Widschwendter et al., 2000), E-cadherin (Nass et al., 2000; Graff et al., 1995; Graff et al., 2000), BRCA1 (Rice et al., 1998; Catteau et al., 1999; Esteller et al., 2000; Rice et al., 2000), HIC-1 (Fuji e tla., 1998), 14.3.3 sigma (Ferguson et al., 2000), HOXA5 (Raman et al., 2000), and TMS1 (Conway et al., 2000).
Allelic loss at the short arm of chromosome 3 is one of the most common and earliest events in the pathogenesis of lung cancer (Witsuba et al., 2000) and is observed in  greater than 90% of small-cell lung cancers (SCLCs) and in 50 to 80% of non-small-cell lung cancers (NSCLCs) (Whang-Peng et al., 1982; Kok et al., 1997). Frequent and early loss of heterozygosity and the presence of homozygous deletions suggest a critical role of the region 3p21.3 in tumorigenesis (Kok et al., 1997; Hung et al., 1995; Wistuba et al., 1999). Loss of heterozygosity (LOH) at 3p21.3 is not limited to lung tumors indicating that this region may harbor a broad-spectrum tumor suppressor gene (Kok et al., 1997). In breast cancer, LOH frequencies have been reported that are in the range of 25-35% using 3p21 markers (Sato et al., 1991; Deng et al., 1994; Matsumoto et al., 1997; Driouch et al., 1998; Braga et al., 1999; Osborne et al., 2000). Frequent loss of heterozygosity and the presence of homozygous deletions suggest a critical role of the region 3p21.3 in tumorigenesis, and recently a region of common homozygous deletion in 3p21.3 was narrowed to 120 kb using several lung cancer and a breast cancer cell line (Sekido et al., 1998). Several putative tumor suppressor genes located at 3p21 have been characterized but none of these genes appears to be significantly altered in lung or breast cancer.
Thus, it is desired to identify a tumor suppressor gene located at chromosme 3p21 which is involved with cancer.
The present invention relates generally to the field of human genetics. Specifically, the present invention relates to methods and materials used to isolate and detect a human tumor suppressor gene, termed RASSF1 herein. More specifically, the invention relates to the analysis of the RASSF1 gene and its use in the diagnosis of predisposition to cancer. The invention also relates to the prophylaxis and/or therapy of cancer associated with the RASSF1 gene. The invention further relates to the screening of drugs for cancer therapy. Finally, the invention relates to the screening of the RASSF1 gene for methylation, loss of heterozygosity or mutations, which are useful for diagnosing the predisposition to cancer. Examples of cancers include, but are not limited to, lung cancer, breast cancer, kidney cancer, ovarian cancer, head and neck cancer and melanoma.
In one aspect of the invention, novel nucleic acids directed to the RASSF1 gene are provided. The nucleotide sequences and corresponding amino acid sequences are set forth as follows: RASSF1.A-SEQ ID Nos:1 and 2, respectively for nucleotide sequence and amino acid sequence; RASSF1.B-SEQ ID Nos:3 and 4, respectively for nucleotide sequence and amino acid sequence; and RASSF1.C-SEQ ID Nos:5 and 6, respectively for nucleotide sequence and amino acid sequence. Novle nucleic acids which hybridize under stringent hybridization conditions to the nucleic acids RASSF1.A, RASSF1.B and RASSF1.C.
In a second aspect of the invention, a method for detecting a susceptibility in an individual to cancer is provided. Thus, the present invention provides methods for determining whether a subject is at risk for developing cancer. This method relies on the fact that methylation of the RASSF1 gene has been correlated by the inventors with many different kinds of cancer. Mutations in the RASSF1 gene have also been discovered and provide an additional basis for establishing a risk for developing cancer. It will be understood by those of skill in the art, given the disclosure of the invention, that a variety of methods may be utilized to detect mutations in the RASSF1 gene, including the mutations disclosed herein, which are associated with a susceptability to cancer. Similarly, a variety of methods may be utilized to detect the presence of a methylated RASSF1 gene or lack of expression of this gene as a result of such methylation.
The method can include detecting, in a tissue or body fluid of the subject, the presence or absence of methylation of the RASSF1 gene. The detection of the methylation may include the detection of methylation, the detection of an aberrant level of mRNA or the detection of an aberrant level of RASSF1 protein.
The method can include detecting, in a tissue of the subject, the presence or absence of a polymorphism of the RASSF1 gene. The detection of a polymorphism in the RASSF1 gene may include ascertaining the existence of at least one of: a deletion of one or more nucleotides; an addition of one or more nucleotides, a substitution of one or more nucleotides; a gross chromosomal rearrangement; an alteration in the level of a messenger RNA transcript; the presence of a non-wild type splicing pattern of a messenger RNA transcript; a non-wild type level of an RASSF1 protein; and/or an aberrant level of an RASSF1 protein.
For example, detecting the polymorphism can include (i) providing a probe/primer comprised of an oligonucleotide which hybridizes to a sense or antisense sequence of an RASSF1 gene or naturally occurring mutants thereof, or 5xe2x80x2 or 3xe2x80x2 flanking sequences naturally associated with an RASSF1 gene; (ii) contacting the probe/primer to an appropriate nucleic acid containing sample; and (iii) detecting, by hybridization of the probe/primer to the nucleic acid, the presence or absence of the polymorphism; e.g. wherein detecting the polymorphism comprises utilizing the probe/primer to determine the nucleotide sequence of an RASSF1 gene and, optionally, of the flanking nucleic acid sequences. For instance, the primer can be employed in a polymerase chain reaction (PCR), in a ligase chain reaction (LCR) or other amplification reactions known to a skilled artisan. In alternate embodiments, the level of an RASSF1 protein is detected in an immunoassay using an antibody which is specifically immunoreactive with the RASSF1 protein.
In a third aspect of the invention, compounds that are agonists of a normal (functional) RASSF1 bioactivity and their use in preventing or treating cancer are provided. For example, to ameliorate disease symptoms involving insufficient expression of an RASSF1 gene and/or inadequate amount of functional RASSF1 bioactivity in a subject, a gene therapeutic (comprising a gene encoding a functional RASSF1 protein) or a protein therapeutic (comprising a functional RASSF1 protein) can be administered to the subject. Alternatively, agonists of RASSF1 function, such as small molecule, peptide, or peptidomimetic, or an RASSF1 receptor can be administered.
In fourth aspect of the invention, assays, e.g., for screening test compounds to identify agonists of the biological function of an RASSF1 protein, are provided. The agonists may bind to and activate a protein or nucleic acid that binds to the RASSF1 protein. An exemplary method includes the steps of (i) combining an RASSF1 protein or bioactive fragments thereof and an RASSF1 target molecule (such as an RASSF1 ligand or nucleic acid), e.g., under conditions wherein the RASSF1 protein and its target molecule are able to interact and measuring the biological acitivity of the target molecule; (ii) combining an RASSF1 target molecule and a test compound under conditions wherein the target molecule and the test compound are able to interact and measuring the biological activity of the RASSF1 target molecule; and (iii) comparing the biological activity of the RASSF1 target molecule in (i) and (ii). The test compound is suitable for preventing or treating cancer if it activates the RASSF1 target molecule.